Histamine is a biological key compound in several organisms, where it serves as a neurotransmitter and hormone. Recent studies showed that it can be metabolized in two ways: by oxidative deamination by diamine oxidase (DAO) or N-methylation by histamine-N-methyltransferase (HNMT). Whether metabolization is achieved via DAO or HNMT depends on the localization of histamine. While DAO is required for termination of histamine signalling in the periphery, HNMT is responsible for inactivation of the biological compound in the central nervous system and thereby prevents histaminergic receptor desensitization. In this study, the effect of a single amino acid exchanges at position 208 was investigated. The crystal structure of the wild-type enzyme was already published by Horton et al. Furthermore, in patients with intellectual disability an amino acid exchange, resulting in leucine to proline at position 208 was elucidated. In order to understand the role of residue 208 in intellectual disability, site-directed mutagenesis was conducted to introduce an exchange at residue 208 in HNMT leading to an amino acid exchange from leucine to valine, phenylalanine, arginine, lysine and histidine, respectively. Isothermal titration calorimetry (ITC) was applied to determine the binding isotherm and the catalytic activity of HNMT (L208) variants. ITC experiments revealed that a replacement at residue 208 only plays a minor role in SAM binding, whereas catalytic activity is decreased 5-fold for the L208F variant in comparison to the wild-type. Thermal shift assays revealed that the thermostability of L208F and L208R is decreased in comparison to the wild-type. Protein determination of the crystal structure would help to support the hypothesis that L208 variants display lower structural stability due to a different -helical content as suggested by circular dichroism (CD) measurements.